Sixty male C57BL/6 mice weighing 20±2 g were used in the study. The mice were obtained from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (Beijing, China; Certificate of Conformity: SCXK Jing 2004-0001). The animals were maintained in a pathogen-free facility (22±2°C, 55±5% humidity) and a 12-h light/dark cycle with lights on from 07:00 to 19:00 h daily. Food and water were provided ad libitum. All the procedures on treating mice were performed according to the Animal Care Guidelines issued by the Ministry of Science and Technology of China and the Animal Care Committee of Liaoning University of Traditional Chinese Medicine approved the protocols.

LLC cells were obtained from the Department of Immunology, College of Basic Medical Science, China Medical University (Wuhan, China) and maintained in Dulbecco's modified Eagle's medium supplemented with 100 ml/l fetal bovine serum, penicillin (1×10⁵U/l) and streptomycin (100 mg/l) with 5% CO₂ at 37°C in a humidified environment.

Cisplatinum (DDP; lot no. 6120251DB) was obtained from Qilu Pharmaceutical Co., Ltd., Jinan, Shandong, China. FJND consisted of 13 herbal materials, including Radix Bupleuri (10 g), Scutellaria baicalensis Georgi (15 g), Rhizoma Pinelliae (10 g), Ginseng (10 g), Fossilia Ossis Mastodi (25 g), Concha Ostreae (25 g), Psuedobulbus Cremastrae (15 g), Zedoary Rhizoma (15 g), Fritillariae Thunbergii (15 g), Radix Platycodi (15 g), Hedyotis Diffusa Willd (20 g), Indian buead (15 g) and Radix Glycyrrhizae (10 g). All were purchased from the Hospital Pharmacy of AHTCM. FJND was extracted by a routine method which is used in the Key Laboratory of Department of Integrated Traditional Chinese and Western Medicine, Peking University School of Oncology, as reported previously (11–13).

Solid-type LLC was prepared by subcutaneous transplantation of 1×10⁷ cells (0.2 ml) into the armpits of 60 C57BL/6 mice. The tumor volume was determined every two days by direct measurement with calipers and calculated using the formula: [Width² (mm²) × length (mm)]/2. The treatment was initiated while all the tumor volumes were >100 mm³ in ≥40 mice. Forty mice were randomly assigned to four groups (10 animals/group); control (CG), DDP (DG), FJND (FG) and FJND + DDP groups (FDG). A 0.9% NaCl solution (0.4 ml/20 g) was administered intragastrically once daily for two weeks in CG and DG mice, and a 0.9% NaCl solution (0.1 ml/20 g) was administered intraperitoneally once daily for 1, 3 and 5 days in CG and FG mice. DG and FDG mice were provided DDP (0.1 ml, 0.1 mg/20 g) intraperitoneally once daily for 1, 3 and 5 days. FG and FDG mice were administered FJND (0.4 ml, 0.62 g/20 g) intragastrically on the same schedule. The mice were treated daily for two weeks and sacrificed on day 15.

Spontaneous activity in 5 min was observed by the photoelectric counting method and all the mice were weighed every other day. Tumor, thoracic gland and spleen tissues were cut to calculate the inhibitory rate (IR), thoracic gland index and spleen index. The IR was calculated as: [(Average tumor weight in the CG group - average tumor weight in the treatment group) / average tumor weight in the control group] × 100%. The thoracic (spleen) index was calculated as: (Thoracic (spleen) weight / body weight) × 100%.

On day 15, the mice were sacrificed by cervical dislocation, and the spleen and thymus were quickly removed and weighed. The spleen tissues were gently teased to release cells by means of dissecting forceps in phosphate-buffered saline (PBS; pH 7.4). The lymphocytes were isolated using density gradient centrifugation (lymphocyte separation medium). The number of lymphocytes was measured using a Coulter counter. The cell concentration was adjusted to 1×10¹⁰ cells/l and 10 µl fluorescein isothiocyanate-labeled anti-mouse CD4 and CD8 (cat. .no. 11-0041-82 and cat.no. 12-0081-82, respectively; both from eBioscience, San Diego, CA, USA) was added to 100 µl of the cell suspension. After incubation for 30 min at 4°C, the lymphocytes were rinsed three times with 1 ml PBS and centrifuged at 500 × g for 3 min. Subsequently, CD4⁺ and CD8⁺ were analyzed by flow cytometry (BD Biosciences, San Jose, CA, USA).

A portion of tumor tissue was fixed by immersion in 10% buffered formalin (pH 7.4) for 24 h. The fixed tissue was dehydrated in graded ethanol, paraffin-embedded and sectioned at a thickness of 4 µm. The tumor histology of each group was observed under a light microscope.

VEGF protein expression was determined for each group by western blot analysis of the protein extracts obtained from the tumor tissue. The protein concentrations were determined by a bicinchoninic acid protein assay (Pierce Biotechnology, Inc., Rockford, IL, USA). Proteins (30 µg) were separated by 12% SDS-PAGE, transferred to polyvinylidene fluoride and blocked with skimmed milk for 2 h at room temperature and incubated with the primary antibody (1:1,000; rabbit polyclonal antibody; BA0407; Wuhan Boster Biological Technology, Ltd., Wuhan, China) at 4°C overnight. The next day the membranes were incubated with the secondary antibody for 1 h (1:5,000; peroxidase-conjugated AffiniPure goat anti-rabbit IgG (H+L); cat. no. ZB-2301; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China). The targeted proteins were visualized by autoradiography. The grayscale was measured using the software BandScan 4.0 (Glyko, Inc., Novato, CA, USA). The relative densities of VEGF protein verses β-actin were calculated.

The values are presented as the mean ± standard deviation relative to the control values. Statistically significant differences from the control group were identified by one-way analysis of variance for the data. P<0.05 was considered to indicate a statistically significant difference.

The body weights of the DG mice were significantly reduced to 14.5±1.01 from 20±0.54 g compared to 16.8±1.24 from 20±0.87 g for the FDG mice. The DG mice became significantly less active in their cages, assessed by rearing and ambulating, compared to the active FDG mice, and the FG mice were more active than the CG mice (Table I).

Tumor growth and final tumor weight were significantly inhibited in the FDG mice. FJND significantly reduced the tumor compared to the CG mice. FJND may have enhanced the antitumor effects of cisplatin. FJND had a synergistic effect with cisplatin in the treatment of LLC-bearing mice (Table II). The spleen weights of FG mice were greater than those of the DG mice. The number of lymphocytes in the spleens of FG mice significantly increased compared to the CG mice. In DG, the number of lymphocytes in the spleens was significantly reduced compared to the FDG mice. However, FJND had no effect on thymus weight.

Following hematoxylin-eosin staining, the change of tumor structure was observed under a light microscope (Fig. 1). Tumor cells grew more productively, proliferated markedly atypical and had a deeper nuclear stain in the CG mice, as compared to the FG mice. In the tumor tissue of the FDG mice, there was a large necrosis area, and a scattered and abnormal small necrosis area. The plasmocytes and lymphocytes infiltrated with scattering or gathering around the necrosis area. The number of plasmocytes and lymphocytes in the DG mice were less in comparison to the FDG mice.

In the FG mice, the splenic CD4⁺ cells and CD4⁺/CD8⁺ were significantly increased compared to CG mice, and in the FDG mice were significantly increased compared to DG mice (Table III).

The protein expression of VEGF significantly downregulated in carcinoma of the FNJD mice compared to the CG mice. The expression of VEGF was significantly reduced in the FDG mice than DG mice (Fig. 2).